The MELT System is a hands-free technology for the rapid digestion of fresh or frozen tissue. No homogenization is required. Samples can be lysed easily in a closed-tube format without cross-contamination. Up to 10 mg of tissue can be digested at room temperature using a novel formulation that includes a potent RNase inhibitor and a cocktail of powerful catabolic enzymes. Following digestion, the RNA is purified using a streamlined process based on Ambion’s patent pending MagMAX magnetic bead procedure and a special vortex adapter. RNase-free DNase is provided to degrade genomic DNA, and the RNA can be eluted from the beads in as little as 20 µl.

RNA Preservation

A major threat to RNA stability in tissues is RNase activity (see RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control). Unlike harsh chaotropes such as guanidine, the MELT reagents digest tissue to the molecular level, reducing problematic RNases to peptide fragments that can no longer degrade RNA. Once digestion is complete, the RNA is highly stable and can be stored at room temperature for more than one week without a significant loss in RNA stability. As shown in Figure 1, a comparison of the MELT System to a common guanidine (GITC)-based lysis method reveals that intact RNA can be purified from MELT lysates even after 10 days of storage at room temperature (22–25°C). Unlike RNA isolated with the MELT System, RNA in the guanidine-based lysate was highly degraded within three days of room temperature storage.

Figure 1. RNA Preservation with the MELT™ Total RNA Isolation System. MELT-liver and GITC-liver lysates were incubated at room temperature (22–25ºC) over 10 days. A fraction of the lysate was removed, and the RNA purified at the time points noted. Agilent® 2100 bioanalyzer gel images of 28S and 18S rRNA bands demonstrate intact RNA isolated from MELT lysates stored at room temperature for several days. MELT=5 mg of frozen mouse liver digested with the MELT System. GITC=5 mg of frozen mouse liver disrupted in guanidinium isothiocyanate-based buffer. ND=Not Determined: Agilent bioanalyzer software unable to define 28S and 18S rRNA peak areas for RIN or ratio quantitation. RIN=RNA Integrity Number, which is based on the entire Agilent electropherogram (range: 0=highly degraded to 10=highly intact).

High Quality MELT RNA is Suitable for Many Applications

RNA isolated from fresh or flash-frozen tissues with the MELT System is appropriate for both quantitative and endpoint RT-PCR. Following total RNA isolation and purification, cDNA products as long as 8 kb can be amplified (Figure 2). This system is ideal for mouse and human studies, because <2% of all known mouse and human transcripts are longer than 8 kb (Dr. Xiaowei Wang, Ambion, personal communication). A comparison of the MELT System with another manufacturer’s kit (Kit 1: guanidinium-based procedure) reveals that amplicons of 0.5, 1.2, 5.0, and 8.0 kb length can be successfully synthesized from RNA purified using the MELT System, which indicates that long, intact transcripts are represented in MELT RNA. In contrast, the 5.0 and 8.0 kb products were much less pronounced using RNA isolated with Kit 1.

Figure 2. Amplicons as Long as 8 kb Effectively Synthesized from Total RNA Isolated with MELT™ Total RNA Isolation System. Total RNA was isolated from ~7 mg of freshly collected mouse liver and brain tissues using 2 different methods: guanidinium isothiocyanate + glass fiber filter-based procedure (Kit 1-GITC) or the MELT Total RNA Isolation System. Total RNA (1 µg) was reverse transcribed using Ambion’s ArrayScript™ RT and oligo(dT) for 1 h at 42ºC. The RT step was followed by 40 PCR cycles using a hotstart Taq polymerase. All RT minus controls were negative for the specific products. Actb=b-actin, Casp3=Caspase-3, PKcs=Protein Kinase C, Fasn=Fatty Acid Synthase.

Each kit contains sufficient reagents to process 50 samples (<10 mg each). A specially designed vortex adapter and either 6 tube or 96 well magnetic stands are available separately for multi-sample processing. The ability of the MELT system to achieve both tissue digestion and RNA preservation enables samples to be safely processed, conveniently stored, and readily transported at ambient temperatures for a host of downstream analyses.